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Honors Theses Division of Undergraduate Studies

2012

The Acute Effects of Late Evening Whey andCasein Ingestion on Fasting Blood Glucose, BloodLipid Profile, Resting Metabolic Rate, and Hungerin Overweight and Obese IndividualsCharles BlayFlorida State University, cjb08c@my.fsu.edu

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Recommended CitationBlay, Charles, "The Acute Effects of Late Evening Whey and Casein Ingestion on Fasting Blood Glucose, Blood Lipid Profile, RestingMetabolic Rate, and Hunger in Overweight and Obese Individuals" (2012). Honors Theses. Paper 101.http://diginole.lib.fsu.edu/uhm/101

Abstract

Background: Theoretically, protein ingestion before sleep should affect obesity rates and

promote cardiovascular health by increasing nocturnal metabolism and decreasing morning

hunger. However, there is little research linking nighttime protein ingestion and morning

cardiovascular health, metabolism, and hunger.

Purpose: To evaluate the acute effectiveness of evening (before sleep) consumption of whey

protein (WP) and casein protein (CP) on improving blood glucose, blood lipids, resting

metabolic rate, and hunger in overweight and obese individuals.

Methods: Forty (n=40; 5 men; 35 women) overweight or obese (age, 28.9 ± 6.6 years; height,

166.2 ± 8.8 cm; weight, 99.0 ± 20.2 kg; body mass index (BMI), 35.7 ± 5.9 kg/m2; % body fat,

46.0 ± 5.8 %) participated in this double blind, placebo-controlled study. Resting baseline

measures of glucose (GLU), total cholesterol (TC), triglycerides (TRG), TC/HDL, high-density

lipoproteins (HDL), low-density lipoproteins (LDL), non-high density lipoproteins (Non-HDL;

TC-HDL), resting metabolic rate (RMR), respiratory quotient (RQ), and a hunger-satiety visual

analogue scale (VAS) were taken in a fasted state after ~8 hours of sleep. Participants were

randomly assigned to WP, CP, or a carbohydrate placebo (PL) supplement to consume before

bed. Participants returned to the lab in a fasted state to repeat baseline measures the next morning

under identical conditions (6 to 8 am).

Results: No significant group by time differences were measured for any dependent variable.

Group differences were measured for GLU to higher and HDL to be lower for CP compared to

both WP and PL. In addition, RMR was elevated to a greater extent for WP and CP compared to

PL, although the difference was not significant. Improvements in hunger, satiety, and desire to

eat were observed from baseline to acute; however there are no group differences.

Conclusion: No significant differences were measured among or between groups for our

dependent variables. However, there was a greater magnitude of change in RMR for WP and CP

compared to PL. Furthermore, the late evening ingestion of WP, CP, and PL before bed

improved morning hunger and satiety after ~8 hours of sleep.

THE FLORIDA STATE UNIVERSITY

COLLEGE OF HUMAN SCIENCES

THE ACUTE EFFECTS OF LATE EVENING WHEY AND CASEIN INGESTION ON

FASTING BLOOD GLUCOSE, BLOOD LIPID PROFILE, RESTING METABOLIC RATE,

AND HUNGER IN OVERWEIGHT AND OBESE INDIVIDUALS

By

CHARLES J. BLAY

A Thesis submitted to the

Department of Nutrition, Food, and Exercise Science

in partial fulfillment of the requirements for graduation with

Honors in the Major

Degree Awarded:

Spring, 2012

The members of the Defense Committee approve the thesis of Charles J. Blay defended

on April 16th

, 2012.

______________________________

Assitant Professor, Dr. Michael J. Ormsbee

Thesis Director

______________________________

Dean of Undergraduate Studies, Dr. Karen Laughlin

Outside Committee Member

______________________________

Associate Professor, Dr. Arturo Figueroa

Committee Member

Acknowledgements

Without the cumulative efforts of the following people, this undergraduate thesis project

would have not been possible to complete. First and foremost, I would like to thank Dr. Michael

J. Ormsbee, my undergraduate thesis director, for allowing me to take part in the acute phase of

his research project, as well as his guidance in concise scientific writing and data analysis. I

would also like to thank my other two committee members, Dr. Arturo Figueroa and Dean Karen

Laughlin of Undergraduate Studies, for their efforts in directing my project. This project

certainly would not have run so efficiently if not for graduate students Amber W. Kinsey and

Wyatt R. Eddy who recruited, scheduled, and conducted an immense portion of the project. Also

graduate student Takudzwa A. Madzima and his insight in the field of acute protein ingestion. I

would also like to thank the undergraduate student workers like Bruce Lee, Emily Mattei, Kelly

Knoth, and Yasmine Kahok. A sincere thanks to our dedicated participants involved in this study

and the Florida State University Council on Research and Creativity for their support of this

research project.

TABLE OF CONTENTS

Section Page number

Review of Literature Table of Contents ..........................................................................................6

Review of Literature .......................................................................................................................7

Introduction ...................................................................................................................................17

Methods .........................................................................................................................................18

Results ...........................................................................................................................................20

Discussion .....................................................................................................................................21

Conclusion ....................................................................................................................................25

References .....................................................................................................................................27

Figures and Tables……………………………………………………………………………….31

Appendices

Appendix A: Institutional Approved Informed Consent ………………………..………35

Appendix B: Health and Fitness History Questionnaire ………………………………...40

Appendix C: Visual Analogue Scale: Hunger/Satiety/Desire to Eat ……………………44

Appendix D: Descriptives, Measurements, and Cholestech Data Sheet …………….….45

Appendix E: Supplement Compliance………………………………………..………….46

Review of Literature

Table of Contents Section Page Number

Overweight and Obesity ………………………………………..………………..……………….6

Evening Food Ingestion …………………..……………..…..………………..………………..…8

Whey and Casein ……………….……………..……………………..………………..………….9

Insulin and Blood Glucose ………………………..………………..……………........……9

Blood Lipids ...……….…..……………………………..…………….................……….10

Blood Pressure …………………....………………..………………..………………..…12

Resting Metabolic Rate …..…………………..………………..………………..……….13

Appetite Suppression …………………………..…………..………………..…..…....….14

Conclusion ………………..………………..………………..………………..…………………15

Review of Literature

Overweight and Obesity

The United States has a public health crisis due to the growth of obesity. National

surveys, like that conducted by the National Health and Nutrition Examination Study

(NHANES) (9), have revealed the prevalence of obesity continuously increasing over the last

three decades. Furthermore, there are no indications of this crisis ceasing or slowing down (43).

Wang et al. analyzed the NHANES data from the 1970s to 2004 for rising

overweight/obesity trends (44). If these trends continued until 2030, 86.3% of the adult

population would be overweight or obese (Body Mass Index (BMI) > 25) with 51.1% of the

population considered obese (BMI > 30). By 2048 all adult Americans would be overweight or

obese. Obesity is not only a health problem in the United States, but it causes severe economic

strain as well. In fact, it is estimated that U.S. health-care costs attributable to the increases in

obesity/overweight prevalence would reach 860.7–956.9 billion US dollars by the year 2030,

accounting for 16–18% of total US health-care costs (44).

The rising prevalence of obesity in the U.S. is a problem due to the widespread associated

health concerns. In fact, it has been suggested that obesity, including excess visceral fat tissue, is

linked to a greater likelihood of cardiovascular disease (CVD) and death (19). Many obese

individuals are susceptible to developing dyslipidemia, type II diabetes, and hypertension. As

visceral obesity increases, factors for metabolic syndrome (MetS) and CVD increase as well.

Scaglione et al. (37) reviewed the known impact of obesity on health components such as

blood lipids, blood glucose, blood pressure, and fat metabolism. Obesity adversely affects

several components of blood lipids. The visceral adipose tissue acts as a source of free fatty acids

(FFA) formed by the hydrolysis of triglycerides. An excessive quantity of FFA in the liver

induces an increased synthesis of triglycerides and promotes the production of very low-density

lipoprotein (VLDL). High triglyceride concentrations are correlated to increased high-density

lipoprotein (HDL) clearance. With less HDL circulating in the blood, there is a decrease in the

cholesterol removing action of HDL and, thereby, atherosclerosis may increase. Atherosclerosis

is also promoted with the hydrolysis of triglycerides into FFA to produce low-density lipoprotein

(LDL) (37).

Excessive FFA concentrations in the blood also induce hepatic gluconeogenesis leading

to hyperglycemia. Furthermore, excessive FFA concentrations may increase insulin resistance in

the muscles by interfering with the intracellular signaling (37). It is likely that a reduction in the

muscles’ sensitivity to insulin may raise blood glucose levels even further, and exacerbate

hyperglycemia.

Hypertension is also associated with having excess adiposity (22). The mechanism

responsible for obesity hypertension is multi-faceted, however, accumulation of visceral fat and

elevated serum insulin and glucose concentrations are thought to be implicated. In addition,

increased renal reabsorption of sodium as a result of these metabolic and hormonal perturbations

may increase blood volume and ultimately, raises blood pressure (7).

Chronic decreases in overall metabolism are expressed through MetS and its underlying

components including dyslipidemia, hyperglycemia, and hypertension. MetS can be a fatal

consequence of obesity. With increasing age and obesity, the components of MetS worsen and

the vicious cycle of obesity continues (37).

Evening Food Ingestion

Late evening ingestion of food is often thought to increase the likelihood of weight gain.

For this reason it is recommended to limit caloric intake in the evening hours because metabolic

rate decreases during sleep (20). Food intake prior to sleep is not metabolized for energy as much

as during earlier times in the day. As a result, more of the food intake is sent towards storage,

rather than being used to provide energy, and leads to weight gain and body composition changes

(20). Postprandial hyperglycemia is often related to late evening food ingestion because glucose

tolerance decreases during sleep (41).

On the other hand, a cross sectional study with non-diabetic late night eaters reported that

ad libitum trials included more kilocalories (kcal) per day than the controlled diet trials (17).

They reported that weight gain is likely due to excessive kcal intake and not to the late evening

meal (25).

Night eating syndrome (NES) is frequently described in obese patients (49). The

prevalence of obese patients seeking weight loss treatment is 6-14%. An even greater prevalence

(from 51 to 64%) has been reported among patients with severe obesity that has been resistant to

any treatment. The concurrent appearance of NES and obesity is also evident because the

presence of obese and overweight NES patients is 57.1% and 28.6%, respectively (49).

Whey and Casein Protein

Whey originates from soluble portions that are removed during the formation of cheeses.

Casein’s origins are from the solid micelles of curd when skim milk is exposed to a low pH (34).

The acute differences in the levels of plasma amino acids from whey and casein ingestion

are due to the differences in the rate of gastric emptying between the two protein sources.

Clotting of casein in the stomach appears to delay its digestion. Due to the quick movement of

whey protein from the stomach to the duodenum (26), large amounts of amino acids are absorbed

within a shorter window of time, resulting in amino acid concentrations much higher than that of

casein. It is thought that this mechanism is responsible for whey’s greater stimulation of muscle

protein synthesis (MPS) compared to casein and other protein types (26).

Borie et al. (8) studied the postprandial differences in plasma amino acid content in

sixteen average male and female young adults using intrinsically labeled 13

C leucine within whey

and casein samples. After ingesting whey, plasma amino acid concentration rose quicker and to a

greater extent at 100 minutes than after ingesting casein. However, the effects from the casein

ingestion were prolonged over a period of 300 minutes. Although whey ingestion created an

early leucine spike, it returned to basal levels after 100 minutes, while casein ingestion created

better overall leucine balance (8).

Insulin and blood glucose

Hoppe et al. (18) examined the effect of milk proteins, whey and casein, on insulin

secretion and blood glucose. These authors reported that whey increased fasting insulin

significantly more than casein. Also, insulin resistance and pancreatic beta cell function were

significantly increased in the whey group, and not in the casein group. The greater content of

BCAAs, leucine, and isoleucine, in whey seem to be the main stimulus for increased insulin

release compared to casein (30). The effect of whey ingestion appears to be dose-dependent

because after acute consumption of various amounts of whey protein, ingestion of more than 20

grams led to increased insulin concentrations; lower blood glucose more than 5 and 10 gram

doses (35). With the prevalence of Type II diabetes increasing, this alternate means of lowering

blood glucose provides a plausible and cost-effective means of protecting and improving overall

health.

Blood Lipids

Total cholesterol (TC) is an extremely important blood lipid measurement derived by the

sum of LDL and HDL. The National Cholesterol Education Program recommends that your TC

levels should not surpass 200 mg/dL (29). Once TC exceeds this number, the likelihood of

coronary heart disease (CHD) greatly increases. Cholesterol promotes atherosclerosis by

building up a plaque on the damaged artery wall and decreasing lumen diameter (22).

Triglycerides are broken down into free fatty acids (FFA) and monoglycerides in the

lumen of the small intestine by pancreatic lipase. They are then absorbed into the enterocytes and

are packaged into chylomicrons to move to the liver. Within the liver, the liposomes form

lipoproteins like low-density lipoprotein (LDL), which is used to carry cholesterol throughout

the body because it is insoluble in the blood. High-density lipoprotein (HDL) is used to collect

cholesterol throughout the body and return it for degradation and excretion in the liver.

Pal et al. (33) studied the long-term effect of chronic ingestion of milk proteins on blood

lipids in eighty-nine overweight and obese individuals between the ages of 18–65 years. Their

finding suggest that fasting triglyceride (TRG) concentrations decreased in the WP group by

13% and 22% after 6 weeks and 12 weeks of whey protein supplementation. Low-density

lipoprotein (LDL) plasma levels were reduced at week 12 in the whey group by 7% compared

with baseline. Similar reductions were seen when compared to casein and the control groups.

Total cholesterol (TC) plasma levels were decreased at week 6 in the whey group when

compared to the control. After 12 weeks, WP comparatively decreased in plasma TC levels by

7% to baseline, 9% to CP and 11% to control (33). Whey protein inhibits the formation of new

cholesterol in the liver (48) and inhibits the expression of genes involved in intestinal FFA and

cholesterol absorption and synthesis (10).

There were no significant changes in body composition after chronic ingestion of whey

protein in 70 middle-aged overweight and obese men and women during a 12-week trial (33).

Beneficial changes in TC, LDL, and TRG from whey supplementation must have been unaided

by changes in body fat mass (33). A meta-analysis by Baigent et al (4) reported that a reduction

in just 1 mmol per L of blood of LDL cholesterol resulted in a decrease of 19% in coronary

mortality. Reductions of TRG levels of 20–24% have also been shown to reduce the progression

of CHD (27).

Blood Pressure

Blood pressure is the most common predictor of future onset of CVD. Increased systolic

blood pressure (SBP) in hypertensive subjects is likely caused by increases in arterial stiffness.

This stiffness causes less cushion in the arteries and a faster pulse wave velocity (PWV) of the

ejected blood. This causes higher left ventricle afterload as the heart contracts during the systole.

Altogether, stiffness in the arteries, which is predicative of CVD, corresponds to the higher SBP

(38).

Blood pressure regulation begins with the renin–angiotensin system and is often

manipulated for hypotensive medication. Supplements that can inhibit the renin–angiotensin

system can be used to treat hypertension (11). This can be accomplished by inhibiting

angiotensin-converting enzyme (ACE) or by blocking angiotensin (AT1) receptors. Previous

evidence indicates that dairy milk proteins (whey and casein) inhibit ACE activity (14, 21, 39)

and in vitro studies specifically indicate that whey has an anti-hypertensive effect (14, 21).

Previous research indicates that whey and casein contain peptides that inhibit ACE

activity (13, 14). Casein and whey degradation produces casokinins and lactokinins, respectively,

which inhibit ACE (13). Both casokinins and lactokinins have been shown to greatly reduce BP,

specifically reductions in systolic blood pressure from 2 to 34 mmHg in both normotensive and

hypertensive individuals (1, 31).

Pal et al. (32) demonstrated that 6-hour postprandial blood pressure (BP) and arterial

stiffness did not have significant reduction with ingestion of 45 g whey protein when compared

with 45 g casein and 45 g of a glucose control in overweight and obese postmenopausal women.

They concluded that the expected hypotensive effects and improvement in measures of arterial

stiffness from whey ingestion are likely only observed over a greater period of time. They did,

however, mention that these unexpected results might be due to the test meal. Consumption of

meal with the supplements likely slowed down the rate of gastric emptying, which could have

delayed or inactivated bioactive components once they reached the small intestine. This likely

reduced the positive effects of whey on BP and vascular function (32). In the present study, we

will investigate the acute effect of consumption of whey and casein alone to avoid any

confounding influences on our results.

Kawase et al. (21) studied the impact of whey protein ingestion on BP on twenty healthy

male volunteers. They reported that after 8 weeks of milk ingestion enriched with whey protein,

systolic blood pressure (SBP) was significantly reduced (21). A similar study showed that 12

weeks of chronic ingestion of whey (54 grams/day) improved arterial stiffness when compared

with casein and a glucose control. Also, both whey and casein reduced diastolic blood pressure

(DBP) when compared with the control after 12 weeks. This implies that a higher dose of whey

and, possibly, a longer duration of supplementation is required for observable effects (33). The

remaining questions to answer are the acute effect of whey and casein on BP without a meal test

and the effect of nighttime ingestion of whey, casein, and carbohydrate on the dependant

variables.

Resting Metabolic Rate

By increasing metabolic rate, our bodies become more efficient in utilizing fat stores,

which then leads a decreased fat mass. Decreased fat mass has already been shown to have

serious health implications, especially when applied to overweight and obese individuals. Not

only does protein deter fat accumulation through satiety, but also it may increase our utilization

of fat stores for fuel.

Acheson et al. (2) studied the differences between macronutrients on 23 healthy lean

participants. Energy expenditure increased after test meals of whey, casein, and carbohydrate.

Expectedly, the thermic effect of the milk proteins was greater than that of the carbohydrate test

meal and the thermic effect of whey was greater than that of casein (34 compared to 24 kcal

increase) (2). These results were conclusively attributed to a greater thermic response and fat

oxidation (2). The previous concerns with BP, blood lipids, and blood glucose are all affected by

visceral fat accumulation. Metabolic efficiency compounded with the acute effect of protein

ingestion could have even greater positive health benefits long-term.

Appetite Suppression

A high protein diet appears to play a role in body weight control because of protein’s

impact on decreasing hunger and increasing satiety (35,47). In fact, Weigle et al. reported that an

isocaloric high-protein (30% DV) diet was able to reduce ad libitum total food intake in 19 men

and women during a 2 week trial (45). This satiating effect may be due in part to greater

secretion of glucagon-like peptide 1 (GLP-1) (16) and cholecystokinin (CCK) (24). GLP-1 and

CCK are released upon entry of chyme into the small intestine for the purpose of increasing

satiety. It has also been reported that protein is a requirement for the release of CCK into our

blood (24).

A high insulin response after whey protein ingestion is due to the insulinotrophic effect of

whey caused by certain amino acids that have insulinogenic properties (15, 30). GLP-1

stimulates the synthesis of insulin secretion, inhibits glucagon, slows the rate of gastric motility,

and inhibits hunger (12). GLP-1 is stimulated by whey’s inhibition of dipeptidyl peptidase IV

(DPP-IV), which is normally responsible for the breakdown of GLP-1.

There are, however, differences between whey and casein in regards to their effect on

satiety. Whey at breakfast appears to suppress appetite more than casein (42). The ingestion of

whey stimulated a stronger response to insulin and GLP-1. WP breakdown had an elevated

production of the amino acids leucine, lysine, tryptophan, isoleucine, and threonine (42). The

high-energy demands of protein breakdown may also be related to satiety (23, 47). Trytophan

has been suggested to have a direct effect on satiety because it is used as a substrate to synthesize

serotonin, which is a neurotransmitter directly associated with appetite (6). Lysine has also been

shown to decrease food intake in sheep (5). Threonine has been shown to reduce weight gain in

rats when it was added to an already low protein diet (28). It is suggested that differences in

appetite ratings between WP and casein only appear when ingestion of each is within a certain

range of protein intake (42).

Conclusion

Obesity’s prevalence, as well as diseases like CVD and Type II diabetes, is rising

worldwide (33). It is possible that protein consumption may reduce total daily caloric intake,

improve fasting blood lipids and glucose and improve metabolic rate. Therefore, protein

supplementation may help in preventing obesity, reducing onset of CVD, and reducing the

likelihood of Type II Diabetes. The effects of evening protein ingestion on risk factors for these

diseases have been under-researched and warrant investigation. The present study will address

the acute effects of protein ingestion (specifically WP and CP) in the late evening (before sleep)

in an attempt to reverse diet-induced diseases like obesity, Type II diabetes, and CVD.

Introduction

Overweight and obese individuals are classified as a having Body Mass Index (BMI;

weight in kilograms divided by height in centimeters2) between 25-29.9 and greater than 30,

respectively (9). Fat accumulation is of multi-factorial etiology, but a primary cause is calorie

intake beyond our daily caloric expenditure needs. Obesity increases one’s likelihood for

developing life-threatening diseases like coronary artery disease and type II diabetes mellitus

(T2DM), and is the leading preventable cause of death worldwide (33).

In most instances, the harmful impact of obesity is apparent when measuring blood lipids,

blood glucose, blood pressure, and resting metabolic rate. Elevated blood lipids and glucose and

blood pressure indicate increased risk for developing atherosclerosis, heart disease, and T2DM.

In addition, variations in daily resting metabolic rate due to obesity and/or nutritional

manipulation can lead to long-term changes in energy balance and, ultimately, alterations to

body weight and composition. Unfortunately, these detrimental effects of obesity are quite

common given that approximately 70% of the US population is considered overweight or obese

(9). Therefore, appropriate research into dietary interventions to combat this growing trend in

body size and disease is needed.

Obesity’s devastating health effects can be offset by proper nutrient intake and exercise

regimens. Interestingly, high protein diets have been shown to increase satiety and may lower

total caloric intake, particularly if fat calories (9kcal/g) are replaced by protein calories (4kcal/g).

Additionally, high protein diets have been demonstrated to increase energy expenditure (2). It is

quite apparent from the existing evidence that including more protein in the diet will ultimately

be beneficial for an overweight/obese population (3). However, which is the type of consumed

protein and timing of ingestion are the most valuable questions left unanswered to date. WP and

CP have been suggested to have positive health benefits. By our inclusion of both milk proteins,

WP and CP, we plan, not just to compare carbohydrates to proteins, but also to investigate

differences between protein types, which are composed of specific amino acid contents.

Therefore, the purpose of this study is to investigate the acute health implications of

nighttime WP ingestion in comparison to CP and PL on blood lipids and glucose, RMR, and

morning hunger, which are all prognostic of health issues and disease.

Methods

Participants. Forty (5 men; 35 women) overweight or obese (BMI > 25 kg/m

2)

participants (Age, 28.9 ± 6.6 years; Height, 166.2 ± 8.8 cm; Weight, 99.0 ± 20.2 kg; Body Mass

Index (BMI), 35.7 ± 5.9 kg/m2; % body fat, 46.0 ± 5.8) were recruited for this study. Each

participant visited the human performance laboratory (HPL) a total of 2 times.

All participants were informed as to the experimental procedures and sign informed

consent statements and medical history forms in adherence with the human subjects’ guidelines

of The Florida State University and with the current national and international laws and

regulations governing the use of human subjects before any data collection. Exclusion criteria

included uncontrolled hypertension (blood pressure (BP) ≥160/100 mmHg), current use of BP

medications, diagnosed cardiovascular disease, stroke, diabetes, thyroid or kidney dysfunction,

milk allergies, or pregnancy. In addition, heavy smoking (>20 cigarettes per day), ingestion of

cholesterol medication or nutritional supplements (except for a multivitamin), or planned

exercise for more than 2 days per week for more than 40-minutes per session (within the past 6

months) were excluded.

Procedures. (Figure 1) The first visit to the HPL (baseline) included arriving in a fasted

state (no food or drink, except water, for at least 8 hours) between 6 and 8 am in athletic

clothing. Questionnaires regarding mood-state, hunger, and satiety were completed. After

sitting quietly for 5-minutes, participants had their baseline blood pressure (BP) was measured

twice. Resting metabolic rate (RMR) was then measured using indirect calorimetry

(Parvometrics, Sandy, UT). This is a non-invasive test that involves lying down on a padded

table for 30-minutes with a ventilated hood covering the head and torso. Blood was drawn for a

total amount of 20 milliliters from a forearm vein (antecubital space between the upper and

lower arm). The blood samples were analyzed for glucose (GLU), total cholesterol (TC),

triglycerides (TRG), TC/HDL, high-density lipoprotein cholesterol (HDL), low-density

lipoprotein cholesterol (LDL), and non-HDL (TC-HDL) concentrations (mg/dL) utilizing the

CholestechLDX blood analysis system (Hayward, CA). Hunger, satiety, and desire to eat were

then assessed utilizing a Visual Analogue Scale (VAS).

After finishing the baseline visit, participants were then matched for BMI, sex, and

percent body fat and randomly assigned to one of three groups in double-blind fashion: 1) 100%

WP consumption in the late evening before sleep (WP), 2) 100% CP consumption in the late

evening before sleep (CP), or 3) PL consumption in the late evening before sleep (Table 1).

Participants in all 3 groups consumed their respective supplements as the last food or caloric

beverage at night before sleep (taken at least 2 hours after dinner, but no more than 30 minutes

before bed).

The WP supplement contained 30g of WP, 3g of carbohydrate, and 2g of fat for a total of

150 kcals per serving. The CP supplement contained 30g of CP, 3g of carbohydrate, and 1g of fat

for a total of 140 kcals per serving. The PL supplement contained 0g of protein, 33g of

carbohydrate, and 2g of fat for a total of 150 kcals per serving. Other ingredients included small

amounts sodium, potassium, and calcium for consistency and flavoring.

On the morning after nighttime consumption of the supplement (between 6 and 8 am),

participants visited the laboratory in the fasted state for the second time (24 hours after visit 1).

Participants were asked to eat the same foods prior to each testing day, with the exception of the

evening supplement to minimize a nutritional influence on the results other than the supplement

consumed. Participants were asked to bring the empty packages to ensure they complied with

protocol and ingested their supplement. The identical testing procedures took place on visit 2 as

were measured on visit 1 to test how acute ingestion with WP, CP, and PL supplements impacted

our dependent variables.

Statistics. A one-way ANOVA was conducted to ensure no differences in groups for BMI

and percent body fat prior to randomization of groups. Data was analyzed using a 3 x 2 (group x

time) RMANOVA (JMP Pro 9, Cary, NC). A Tukey post-hoc analysis was used where

appropriate to examine differences. Significance was set as P<0.05 and all data are reported as

means ± SD, unless otherwise noted.

Results

Blood glucose and lipids

No significant group by time differences were measured for glucose (GLU), total cholesterol

(TC), triglycerides (TRG), TC/HDL, high-density lipoproteins (HDL), low-density lipoproteins

(LDL), or non-high density lipoproteins (Non-HDL; TC-HDL). Group differences were measured

for GLU to higher and HDL to be lower for CP compared to both WP and PL. (Table 2).

Resting Metabolic Rate (RMR)

No significant group by time differences were observed for RMR or respiratory quotient (RQ),

although there was a magnitude change from baseline to acute for all groups. There was a main

group effect (p=0.02), which post-hoc analysis revealed there to be a significant difference

between WP (1941.2 ± 187.5 kcal) and CP (2150.2 ± 288.6 kcal), but not PL (2035.0 ± 211.4

kcal) (Figure 2).

Hunger, Satiety, Desire to Eat

There was a significant time difference from baseline to acute in all three groups, but no group or

group by time differences (Figure 3).

Discussion

This study investigated the immediate health implications of nighttime WP ingestion in

comparison to CP and PL on GLU, blood lipids, RMR, and morning hunger, which are all

prognostic of health issues and disease.

Increased protein consumption has been reported to have various acute and long-term

health benefits including improvements in GLU, blood lipids, RMR, and satiety (33, 2, 40, 45).

In addition, protein consumed at particular times of the day has become a topic of much interest

(3, 36). However, few reports have examined the impact that protein supplementation has on

cardiometabolic health when consumed before sleep. Furthermore, only one study to date (36)

has linked protein ingestion before bed to improvements in muscle protein synthesis, and none

link nighttime protein ingestion and morning cardiovascular health, metabolism, and satiety,

despite the common thought that eating before bed may be harmful to health. By our inclusion of

both milk proteins, WP and CP, we planned, not just to compare carbohydrates and proteins, but

also to investigate two protein types which differ in amino acid composition. When comparing

these two proteins, it is important to understand the differences in digestion of these two

macronutrients. WP has been shown to more quickly raise plasma amino acid concentration

when compared to CP due to the differences in the rate of gastric emptying. CP clots more in the

stomach, which delays digestion while WP rapidly moves from the stomach to the duodenum

(26). Due to this quick movement of WP, large amounts of amino acids are absorbed within a

shorter window of time, resulting in amino acid concentrations much higher than that of CP.

Acute differences in plasma amino acids content may accentuate other cardiometabolic

differences between WP and CP.

Our primary findings reveal no statistically significant group by time interactions

between WP, CP, and a PL in terms of fasting GLU, blood lipids, and RMR after evening

ingestion. Despite the lack of significance, it is important to note that WP increased RMR by 4.5

± 0.3% and CP increased RMR by 2.7 ± 0.3%, while PL decreased RMR 2.6 ± 0.3%, which

could have practical implications In addition, significant time effects between baseline and acute

were observed for all 3 groups to improve scores of hunger, with no difference between groups.

Our baseline-to-acute change in fasting GLU was not statistically significant, which

agrees with a previous 7-day intervention study using WP and CP with prepubertal boys (WP:

baseline 4.52±0.38, post 4.58±0.29; CP: baseline 4.47±0.26, post 4.53±0.24 mmol/L) (18). These

effects on fasting GLU appear to be affected by the specific amount of macronutrient ingested

and are dose-dependent (35). While we report no differences in GLU, it would be interesting to

evaluate the different levels of specific branched-chain amino acids, leucine and isoleucine,

within the blood, which appear to be proportional to blood insulin levels (30). This is especially

appropriate considering the higher content of these specific amino acids in WP (isoleucine:

6.20%; leucine: 10.40%) compared to CP (isoleucine: 4.89%; leucine: 9.41%). Additionally,

muscle insulin sensitivity may have been affected by ingesting the supplements in the present

study. Ingestion of WP for 7 days, as opposed to CP, has been shown to significantly increase

insulin concentration (WP: baseline 33.00±11.6, post 39.93±14.5 pmol/L; CP: baseline

37.17±12.4, post 40.90±23.9 pmol/L) and increase insulin resistance using the homeostatic

model of insulin resistance (HOMA) (WP: baseline 1.12±0.42, post 1.37±0.52; CP: baseline

1.25±0.46, post 1.39±0.86). This increase in insulin concentration and resistance occurs in

proportion to one another, which is likely why hyperinsulinemia may not be recognized by GLU

measurements alone (18). While we can only speculate these facts may coincide in the present

study, it is possible that differences between groups may have existed in insulin concentrations.

Raising insulin concentration will improve GLU only if the proportional rise in insulin resistance

were combated with resistance training.

We measured no statistically significant changes in fasting blood lipids: TC, TRG,

TC/HDL, HDL, LDL and non-HDL. Previous research has shown decreases in TRGs and LDLs

over the course of 6 and 12 weeks with supplementation of whey in comparison to a

carbohydrate placebo (33). These improvements are of significance for cardiometabolic health.

TRG and LDL improvements were not associated, however, with decreases in fat mass. The

divergence between our research and that of Pal et al (33) is likely due to the duration of each

supplement intervention. Long-term improvements in blood lipid concentration by WP and CP

ingestion, as well as the compounded effect of improvement in body fat percentage should

improve cardiometabolic health over a longer period of time.

No significant changes were observed in RMR as a result of WP, CP or PL

supplementation. However, WP increased RMR by 4.5 ± 0.3% and CP increased RMR by 2.7 ±

0.3%, while PL decreased RMR -2.6 ± 0.3%. These changes, albeit not significant, represent

approximately a ~140 kcal increase in daily RMR when comparing WP to PL. Extrapolating this

to a full week, nighttime protein consumption could result in a ~980 kcal increase in metabolism.

These positive results are consistent with Acheson et al. who also reported the greatest

improvement in morning RMR in WP (14.4±0.5%), followed by CP (12.0±0.6%), and PL

(6.6±0.5%) during a 5-treatment (baseline measured before each morning ingestion of given

supplement), diet-controlled intervention of healthy individuals (2). These increases are expected

due to the complexity of proteins in comparison to simple sugars, which requires a greater

amount of energy for their metabolism. The long-term practical effect of a higher protein diet

may be important for decreasing fat mass and improving body composition by a consistently

elevated metabolic rate.

We utilized the Visual Analogue Scale (VAS) to assess fasting hunger, satiety, and desire

to eat after nocturnal sleep at baseline and following consumption of WP, CP, or PL. Other

research has suggested through ad libitum trials that protein, WP in particular, decreases hunger

and desire to eat by increasing satiety (40, 45). This increased feeling of satiety is due to the

increased cholecytokinin (CCK) secretion in response to amino acid-rich chyme in the intestines

(24). Whey is thought to increase amino acid content more so than casein and carbohydrate due

to its high branched-chain amino acid composition (24). Our data reveals that whether

participants ingested WP, CP, or PL they had decreased subjective feelings of hunger, increased

satiety, and a decreased desire to eat the morning after ingestion of the supplement in comparison

to baseline. Thus, participants felt fuller after ingesting kilocalories the night before, regardless

of their composition, which has not previously been documented after an overnight fast. Serum

CCK concentrations and other appetite hormones would likely reveal if any physiological

measures of fullness were different between groups.

Whey and casein protein are known to have acute and long-term effects on

cardiometabolic health, macronutrient metabolism, and dietary decisions after consumption in

both the short-term and long-term. Our data indicate a lack of significance in any of the

dependent variables; however, the benefit of WP and CP consumed before sleep may be shown

to improve health when similar testing is conducted over a longer period of time or in more

participants. Interestingly, the observed non-significant trend for RMR to increase for WP and

CP, and a decrease for PL group may have significant influence on long-term metabolism. These

results, compounded with the resulting increases in morning satiety, may provide a means of

improving overall caloric balance by decreasing ad libitum intake and increasing metabolic rate.

Thus, while no statistically significant findings were observed, there may be practical

implications to our findings.

The study has several limitations that should be addressed. While this study was designed

to test the acute impact of WP and CP on health when consumed in the evening before sleep, we

might have observed different results if the study had been longitudinal in design. We also

studied forty individuals, including men and women. With a larger number of participants as

well as separation of data by sex, we might have found that our non-significant changes were, in

fact, statistically significant. Additionally, we can only speculate as to the mechanisms behind

our findings. Future investigations must include serum measurements of amino acids and insulin

to reveal possible mechanisms of action. Similarly, serum concentrations of leptin and CCK

might better describe the physiological response of evening WP and CP ingestion on morning

hunger and satiety. In addition, we did not include measurement of regular dietary intake data for

our participants, which may influence our primary outcome variables. Also, participant

compliance to supplement ingestion and timing of intake (2 hours post-dinner, 30 minutes before

sleep) may not have been perfect; however, our participants reported 100% compliance with the

supplement intake for this study, which was regulated by the collection of empty supplement

bags at visit 2. Also, we did not control for menstrual cycle phase in the present study and this

should be accounted for in future research. The addition of an exercise regimen may also alter

the physiological response to these macronutrients when consumed at night before bed and round

out a balanced lifestyle intervention designed to improve overall health.

Conclusions

The purpose of this study was to investigate the immediate health implications of

nighttime WP ingestion in comparison to CP and PL on blood GLU, blood lipids, RMR, and

morning hunger, which are all prognostic of health issues and disease. No significant differences

were observed among or between groups for GLU, blood lipids, RMR, and hunger. However,

there was a greater magnitude of improvement for RMR for WP and CP compared to PL.

Furthermore, regardless of macronutrient choice, eating before bed appeared to improve hunger,

satiety, and desire to eat. Thus, protein ingestion before bed may provide a practical health

benefit, although more data is warranted. It is likely that a longer duration study with more

participants and exercise training would reveal significant differences between these groups.

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Figure Headings.

Figure 1: Overall Study Design. Study timeline. All testing was completed in the fasted state.

At baseline, participants had no consumed any evening meal the night before morning testing. At

acute testing, participants had consumed whey, casein, or placebo supplementation the night

before morning testing. Supplement consumption was 2-hr after the last meal and 30 min before

sleep. Overweight and obese individuals participated in this study (n = 40).

Figure 2: Resting Metabolic Rate. Energy expenditure at baseline and the morning after

evening ingestion of whey, casein, or placebo supplements (acute) in 40 overweight and obese

individuals

Figure 3: Hunger, Satiety, and Desire to Eat. Huger, satiety and desire to eat as assessed using

a visual analog scale in 40 overweight and obese individuals at baseline and the morning after

evening ingestion of whey, casein, or placebo supplements (acute). *, p<0.05 compared to

baseline for all groups.

Figure 1

Figure 2

Resting Metabolic Rate

1700

1800

1900

2000

2100

2200

2300

2400Whey

Casein

Placebo

Baseline Acute

kcal

Visit 1

-Metabolic Testing

-Blood Samples

-Hunger and Satiety

Visit 2

-Metabolic Testing

-Blood Samples

-Hunger and Satiety

Hunger

20

30

40

50

60Whey

Casein

Placebo

Baseline Acute

cm

*

Satiety

0

510

15

2025

30

35

4045

50

55Whey

Casein

Placebo

Baseline Acute

cm

*

Desire to Eat

30

35

40

45

50

55

60

65Whey

Casein

Placebo

Baseline Acute

cm *

Figure 3

Table Headings

Table 1: Participant Characteristics. Values are means ± SD. BMI, body mass index; n=

number of subjects.

Table 2: Cardiovascular Measures. Values are means ± SD. GLU, fasting blood glucose; TC,

total cholesterol; TRG, triglycerides; HDL, high-density lipoproteins; LDL, low-density

lipoproteins; Non-HDL, TC-HDL; Placebo, carbohydrate; n = 40. Differences were significant

at P<0.05.

Table 1

Table 2

Whey

(n=15: 2 Males)

Casein

(n=14: 2 Male)

Placebo

(n=11: 1 Male)

Age (year) 27.1 ± 5.9 32.1 ± 7.0 27.1 ± 7.2

Height (cm) 165.4 ± 11.2 167.0 ± 8.6 166.1 ± 5.8

Weight (kg) 96.5 ± 17.7 104.7 ± 24.5 95.0 ± 18.1

BMI (kg/m²) 35.7 ± 4.7 37.5 ± 7.8 34.2 ± 5.2

% Body Fat 45.6 ± 7.3 45.9 ± 6.7 46.8 ± 4.6

Values are Mean ± SD. P>0.05 for all variables.

Whey Casein Placebo

Baseline Acute Baseline Acute Baseline Acute

GLU, mg/dL 85.9 ± 5.4 B

84.4 ± 5.4 B

95.6 ± 5.4 A

94.8 ± 5.4 A

86.0 ± 5.4 B

86.5 ± 5.4 B

TC 169.0 ± 13.1 163.6 ± 13.1 161.3 ± 13.1 158.8 ± 13.1 173.6 ± 13.1 170.4 ± 13.1

TRG 104.6 ± 27.1 89.5 ± 27.1 99.0 ± 27.1 108.6 ± 27.1 99.3 ± 27.1 86.0 ± 27.1

TC/HDL 4.1 ± 5.5 6.9 ± 5.5 4.2 ± 5.2 4.0 ± 5.2 3.8 ± 5.2 3.7 ± 5.2

HDL 40.2 ± 8.2 41.8 ± 8.2 40.1 ± 8.3 42.1 ± 8.3 49.1 ± 8.3 48.5 ± 8.3

LDL 104.8 ± 16.2 105.6 ± 16.2 102.4 ± 14.5 97.3 ± 14.5 107.5 ± 14.1 108.9 ± 14.1

Non-HDL 120.0 ± 17.4 118.9 ± 16.3 121.8 ± 16.3 119.1 ± 16.3 124.3 ± 16.3 121.9 ± 16.3

Values not connected by the same letter are significantly different

The effect of protein timing and combined resistance and high-intensity interval training on body composition, blood lipids, growth hormone, and strength in overweight and obese individuals.

Informed Consent Form 1. I voluntarily and without element of force or coercion, consent to be a participant in the

research project entitled “The effect of protein timing and combined resistance and high

intensity interval training on body composition, blood lipids, growth hormone, and strength in

overweight and obese individuals.” This study is being conducted by Dr. Mike Ormsbee, Dr.

Arturo Figueroa, Dr. Robert Moffatt, Dr. Jeong-Su Kim, Dr. Lynn Panton, Amber Kinsey,

David Thomas, and Wyatt Eddy of the Department of Nutrition, Food & Exercise Sciences at

Florida State University.

2. The purpose of the proposed study is to examine how protein supplementation in the late

evening before sleep and exercise training affect body composition, anabolic and appetite

hormones, fat metabolism, stress, and strength. Sixty sedentary, overweight or obese men

and women (18 to 45 years of age) will be recruited for this study.

3. My participation in this study will require coming to the Human Performance Laboratory at

Florida State University for testing on four different occasions over 4 weeks to complete the

measurements and assessments as described below.

On my first visit, I will be given an informed consent document to sign and a medical history

form to complete before I can participate in the study. I cannot participate in this study if I

have uncontrolled hypertension (Blood Pressure (BP)>160/100 mmHg), take BP

medications, have been diagnosed cardiovascular disease, stroke, diabetes, thyroid or

kidney dysfunction, milk allergies, am pregnant, or have any musculoskeletal complications

(i.e., osteoarthritis or injury) that would impede me from exercising. In addition, I will be

excluded if I am currently a heavy smoker (>20 cigarettes per day), take cholesterol

medication or nutritional supplements (except for a multivitamin), or partake in planned

exercise for more than 2 days per week for more than 40-minutes per session (within the

past 6 months).

During this visit, I will then answer questionnaires regarding physical activity, nutritional

habits, and mood-state. I will have my blood pressure, height, weight, waist and hip

circumferences, body composition, and strength measured. Height and weight will be

assessed using a standardized scale. Waist circumference measures will be taken a

minimum of two times. My body composition and bone mineral density will be measured

using dual energy X-ray absorptiometry (DXA). Very low doses of radiation are used;

however, this test is noninvasive. I will lie on a padded table for approximately 10 minutes

while the scan is being completed. Testing will be completed according to the

manufacturer’s instructions and specifications by a certified X-ray technician. Both upper

and lower body strength will be assessed using the chest press and leg press exercises,

respectively. After a warm-up period, I will be progressed towards the maximum weight that I

can lift 1-time through a full range of motion, also called a 1-repetition maximum (1-RM). All

measurements will be recorded within three and five attempts and will be supervised by

trained personnel.

I will be given food record forms (to list all foods and beverages consumed over 3 days) to

bring filled out on the next visit and will receive instructions on how to complete these forms.

I will get familiarized with the metabolic and cardiovascular testing equipment on this day.

This visit will take approximately 2 hours.

On the second visit (occurring at least 48 hours following the first visit), I will come to the

laboratory in a fasted state (no food or drink, except water for at least 8 hours) between 6

and 11 am. I will turn in the 3 day food record and then have my resting metabolic rate

(RMR) measured using indirect calorimetry. This is a non-invasive test that involves lying

down on a padded table for 30-minutes with a ventilated hood covering my head and torso. I

will have my cardiovascular function evaluated after 20-minutes of rest in the supine (lying

down) position. A total of 4 cuffs, one in each extremity (around arm and ankles) and 2

tonometers (sensors applied to the skin to obtain pulse waves), one on the neck and the

second on the inner thigh, will be used to measure pulse wave velocity (arterial stiffness).

My blood pressure will be also monitored by placing a small cuff around the middle finger

and a tonometer on the wrist and neck. Six electrodes will be positioned on the skin of my

chest to measure heart rate (electrocardiogram). Arm blood flow will be measured using

vascular ultrasound positioned on my skin at rest and during increased blood blow after

deflation of an arm cuff (5 minutes inflation). The diameter and thickness of my neck artery

(common carotid) will be measured non-invasively by ultrasound. I will also have my blood

drawn on 3 occasions under sterile conditions (2 blood draws per each visit) and the total

amount of 20 milliliters from a forearm vein (between the upper and lower arm) and finger

prick and stored for later analysis. The blood samples will not be used for any other

research or testing purposes other than those specified in the research proposal. I will have

my saliva collected by placing a salivary oral swab underneath my tongue for 2-minutes.

The second visit should take approximately 90 minutes.

After finishing visit two, I will be randomly assigned to one of three intervention groups for

the duration of the four-week intervention: 1) 100% whey protein consumption in the late

evening before sleep (WP), 2) 100% casein protein consumption in the late evening before

sleep (CP), or 3) placebo (carbohydrate) consumption in the late evening before sleep

(CON). Participants in all groups will consume their respective supplements as the last food

or caloric beverage consumed prior to going to sleep.

The whey protein supplement will contain 30g of whey protein, 3g of carbohydrate, and 2g

of fat for a total of 150 kcals per serving. The casein protein supplement will contain 30g of

casein protein, 3g of carbohydrate, and 1g of fat for a total of 140 kcals per serving. The

placebo supplement will contain 0g of protein, 33g of carbohydrate, and 2g of fat for a total

of 150 kcals per serving. Other ingredients will include small amounts sodium, potassium,

and calcium for consistency and flavoring.

On the next morning (between 6 and 11 am), for my third visit (24 hours after the second

visit), I will arrive to the human performance laboratory in a fasted state (for at least 8 hours).

I will then have my body weight, resting metabolic rate, and cardiovascular function

measured. I will also have blood and saliva collected and fill out a hunger and mood-state

questionnaire as described above. The third visit will last approximately 90 minutes.

After the third visit, I will continue with my late evening drink consumption as previously

assigned every night of the week (7 nights) and I will complete three workouts (2 resistance

training days, 1 high-intensity interval training day) under the supervision of qualified

instructors each week for four weeks. Each exercise session will last for approximately 45

minutes. Resistance exercises will consist of the following exercises: chest press, seated

row, leg press, shoulder press, leg extension, and leg curl. Each exercise will be performed

for 3 total sets: 2 sets of 10 repetitions and a 3rd set to muscular exhaustion with a load

equaling 70-85% of the individual’s previously established 1- RM. Rest periods will be set to

90-120 seconds between all sets and exercises and the RE session will last for a total of 40

to 45 minutes.

The one cardiovascular training day per week will use a high-intensity interval program in

which participants will rate their perceived exertion on a scale from 1 to 10 (1= resting

quietly, 5= a warm-up level, 10= an all-out exertion). Participants will begin with a 2 minute

warm-up at level 5 and increase their exertion each minute for 3 minutes until level 9 is

perceived and then recover at level 6 for 1 minute. This pattern is repeated four times,

however, on the fourth cycle participants will increase their last minute of exertion to level

10, followed by 1-minute recovery at their initial warm-up level 5. The exercise duration in

total will be 20-minutes. In addition, I will wear a pedometer (step-counter) daily to measure

physical activity over the 4- week study.

I will repeat my 3-day food diary again during the final week of the 4-week training period

and turn it into the research staff. All measurements taken during visits one and two will be

replicated for visit 4 following the 4-week intervention.

4. I understand there is a minimal level of risk involved if I agree to participate in this study. I

may experience some muscle soreness from the 1-RM and exercise training sessions. The

risks associated with 1-RM and exercise training are minimal and the selected protocols

have been previously used in other studies in sedentary men and women. There is the

possibility of muscle fatigue or soreness related with exercise training or testing. Although

there is a potential risk of muscle injury with maximal strength testing (1-RM), the risk will be

reduced by using a submaximal strength test, the 1-RM. The risk will be minimized by using

qualified exercise instructors to supervise testing and training and ensure proper exercise

techniques and intensity. The risk of a cardiovascular event during testing and training will

be minimized by careful review of my medical history and monitoring of my exercise

sessions. In addition, my cardiovascular exercise is based off of my perceived exertion and

is therefore individually tailored to my level of fitness. I understand that to reduce muscle

fatigue and soreness my trainer may make adjustments to my training program. I am aware

that the facility that produces the supplements for this study may also manufacture products

made from soy, wheat, and grain at the facility. It is possible that cross-contamination could

occur, but is unlikely. If I have an allergy to milk, soy, wheat, or grain I must make this known

to the research team.

The risk of blood drawing is small and there may be some local discomfort at the site of

needle placement with possible bruising or swelling. The risk of local infection is also small.

These risks will be minimized by the use of skilled technicians using sterile techniques and

equipment.

Body composition will be evaluated by Dual-Energy X-ray Absorptiometry (DXA). This

involves some radiation of approximately 0.5 mREM per total body scan or 1 mREM for both

scans. This is much less than a traditional chest X-ray (20-50 mREM) or full dental X-ray

(300 mREM). The measurement of body composition using DXA is non-invasive.

5. The possible benefits of my participation in this research project include about my body

composition, bone mineral density, resting vital measures, waist and hip circumferences,

resting metabolic rate, upper and lower body muscular strength, heart rate control and

arterial function. Participants in both groups will have the potential to improve metabolic,

cardiovascular and muscular health and may improve body composition, physical

functioning, and quality of life. I will also be given 12 training sessions at no charge.

6. The results of this study may be published but my name or identity will not be revealed.

Information obtained during the course of the study will remain confidential, to the extent

allowed by law. My name will not appear on any of the results. No individual responses will

be reported. Only group responses will be reported in the publications. Confidentiality will be

maintained by assigning each subject a code number and recording all data by code

number. The only record with the participant’s name and code number will be kept by the

principal investigator, Dr. Michael Ormsbee, in a locked drawer in his office. Data will be

kept for 10 years and then destroyed.

7. In case of an injury, first aid (free of charge) will be provided to me by the laboratory

personnel working on the research project. However, any other treatment or care will be

provided at my expense.

8. Any questions I have concerning the research study or my participation in it, before or after

my consent, will be answered by the investigators or they will refer me to a knowledgeable

source. I understand that I may contact Dr. Michael Ormsbee at (850) 644-4793

(mormsbee@fsu.edu), or Amber Kinsey at awk10d@fsu.edu for answers to questions about

this research study or my rights. Group results will be sent to me upon my request.

9. In case of an injury, or if I have questions about my rights as a subject/participant in this

research, or I feel I have been placed at risk, I can contact the chair of the Human Subjects

Committee, Institutional Review Board, through the office of the Vice President of Research

at (850) 644-8633 (humansubjects@magnet.fsu.edu).

10. The nature, demands, benefits and risks of the study have been explained to me. I

knowingly assume any risk involved.

11. I have read the above informed consent form. I understand that I may withdraw my consent

and discontinue participation at any time without penalty or loss of the benefits to which I

may otherwise be entitled. In signing this consent form, I am not waiving my legal claims,

rights or remedies. A copy of this consent form will be given to me.

________________________________ Print name ________________________________ ________________________________ Signature Date

Human Performance Laboratory Florida State University

Nutrition, Food, and Exercise Sciences

HEALTH AND FITNESS HISTORY QUESTIONNAIRE

The following questions are designed to obtain a thorough preliminary medical history. The information you provide will help us to make the best determination about your eligibility for a particular study or other studies. Please answer all questions and provide as much information as you possibly can. This questionnaire, as well as any other medical information you provide will be kept confidential and will not be shared with any unauthorized person or organization unless you specifically request us to do so. Name:

Street Address:

City, State, Zip code:

Telephone Number: H ( ) W ( )

Email address: _________________________________________________ Date of Birth: Age: (mm/dd/yy)

Sex: M F

Personal Physician’s Name: Phone: ( )

Address:

Height in. ___________ cm

Weight lb. __________ kg

Social Security Number:

Signature:

Date: __________ ID #: __________

Occupation

Current occupation:

Race ______________ Personal Health History

Have you ever been hospitalized or had surgery? Yes____ No____ Please list all hospitalizations and surgeries to the best of your recollection.

Hospitalized for Age when Disease/Operation Duration hospitalized List any disease or illness you have had not listed above (e.g., mumps, measles, broken bones, etc.)

Are you allergic, sensitive or intolerant of any foods or medications? Yes____ No____ If yes, please describe: Food____________________________________________________________ Medication _______________________________________________________ Other ___________________________________________________________ Are you currently seeing a doctor or other health care provider for any reason?

Yes______ No______

If yes, please explain:

Date: __________ ID #: __________

1. Have you ever been diagnosed as having any of the following and if yes, how are you currently treating

the condition?

Y N High Blood Pressure

Please indicate last known reading:

Blood pressure: _____/_____

Y N High Cholesterol or High Triglycerides

Please indicate last known reading:

Cholesterol: _____

Triglycerides: _____

Y N Diabetes (Circle: Type 1 or Type 2)

Note: Type 1 diabetes is insulin-dependent diabetes mellitus. It is typically

diagnosed at an early age and requires insulin shots or an insulin pump

immediately upon diagnosis. Type 2 diabetes is often diagnosed at an

older age (past age 20) and is usually initially treated with changes in diet

and/or medication (pills).

Y N Hypoglycemia (low blood sugar)

Y N Asthma

2. Have you ever had a glucose tolerance test? Y N

If yes, what were the results?

3. Have you ever had a fasting blood sugar test? Y N

If yes, what were the results?

4. Does anyone in your family (immediate family including your grandparents) have a history of

cardiovascular disease (heart attacks, stroke, etc.)? Please explain:

5. Do you have any neurological problems including fainting, dizziness, headaches or seizures?

6. Do you have any orthopedic or other health problems that may affect your ability to perform exercise?

If yes, please explain:

7. Do you smoke or use smokeless tobacco? Y N If yes, how many cigarettes per day? ______

8. Do you drink coffee or other caffeinated beverages? Y N What kind, how much and how

often?

Date: __________ ID #: __________

9. Please list all vitamins, minerals and herbs and other nutritional (performance) supplements as well as

medications you are currently taking. How long have you been taking them and how frequently?

Are you willing to stop taking all nutritional supplements you are currently on for the duration of this

research study? (Y/N) ___________________

10. Do you have any food allergies or intolerances (e.g., allergic to dairy or lactose intolerance)? Please

describe:

11. How would you describe the type of diet you currently eat? Have you recently been on any special

diets? What kinds of diets have you used to lose weight or lower cholesterol? Please list and describe:

12. What changes have you made in your diet in the last 6 months?

13. Do you exercise regularly? Y N What kinds of exercise?

How often? Please be detailed in a description of your average week of training.

Please list the 3 most current athletic events/competitions that you have participated in:

14. How does your current exercise and physical activity compare to 6 months ago? 1 year ago?

15. Have you had a physical exam in the past 2 years? Y N

Please describe your assessment of your overall health:

16. To what extent does snacking after dinner contribute to your weight? Circle one.

1 2 3 4 5

Not at all To a very large ext

ID# Date / /

TEST#: Pre Post

Please indicate the level to which you are feeling ALL three of the following with a mark on

the line:

1) Do you feel HUNGRY:

Not at all Extremely

2) Satiety (feeling of fullness):

Not at all Extremely

3) Desire to eat:

Not at all Extremely

Evening Protein and Exercise Training Study Data Sheet

Subject ID: ________ Age: ______ Date:_________ (circle one): Baseline Post DOB: ________

Women only - Start Date of Menses: ____________ Birth Control: ___________ Heart Rate 1. __________ 2. __________ Height: _______cm _______in Blood Pressure 1. __________ 2. __________ Weight: _______kg _______lbs Waist circumference 1.__________ 2. _________ Cholestech Hip circumference 1. __________ 2._________ Waist:Hip ratio __________

Notes: (ex: clothing worn for circumferences)

TC ______ LDL ______ TRG ______ HDL ______ TC/HDL ______ Non-HDL ______ GLU ______

______ (tech initials)

______ (tech initials)

Supplement Compliance

Evening Protein and Exercise Training DATE: - -

Subject No: Subject Initials: Supplement Reminder

(given - initial/returned - initial) (called - date and initial) Initial ______ ____/______ ____ _____________________ Week 1 ______ ____/______ ____ _____________________ Week 2 ______ ____/______ ____ _____________________ Week 3 ______ ____/______ ____ _____________________ Week 4 ______ ____/______ ____ _____________________